Most evidence indicates that G protein-coupled receptors (GPCRs) can form homo and heteromers. Homodimers seem to be a predominant species with potential dynamic formation of higher-order oligomers. The pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may provide a main functional unit at the plasma membrane and oligomeric entities can be viewed as multiples of dimers. Our recent studies, particularly on the adenosine A2A-dopamine D2 receptor heteromer, indicate that GPCR heteromers are often constituted by heteromers of two homodimers, i.e. heterotetramers (1-3). Allosteric mechanisms determine a multiplicity of possible unique pharmacological properties of GPCR homomers and heteromers. Some general mechanisms seem to apply, particularly at the level of ligand-binding properties, but also unique properties for GPCR heteromers emerge in relation to different intrinsic efficacy of ligands for different signaling pathways (functional selectivity) (1-3). We are interested in GPCR heteromers localized in the brain and, particularly, in brain circuits involved in substance use disorders. We have discovered several heteromers that control striatal function, involving adenosine and dopamine receptors. For instance, the postsynaptic A2A-D2 receptor heteromers localized in the GABAergic striato-pallidal neurons (1-3) and the presynaptic A1-A2A and A2A-cannabinoid CB1 receptor heteromers localized in cortico-striatal glutamatergic terminals (4,5). We have also been interested in heteromers of the dopamine D1 receptor, localized in the GABAergic striato-nigral neuron, more recently in D1-ghrelin receptor heteromers (6). Finally, we have also been interested in D2-like receptor heteromers also localized in glutamatergic terminals, particularly those including dopamine D4 receptors and their enigmatic polymorphic variants (7). In relation to the striatal A2A receptor heteromers, first, we have introduced a model that considers the A2A-D2 receptor heterotetramer as a key modulator of dopamine-dependent striatal functions (reward-oriented behavior and learning of stimulus-reward and reward-response associations) (5). The striatal A2A-D2 receptor heterotetramer constitutes an unequivocal main pharmacological target of caffeine and provides the main mechanisms by which caffeine potentiates the acute and long-term effects of prototypical psychostimulants (3). To study A2A receptor heteromers localized presynaptically in cortico-striatal glutamatergic terminals, we have introduced a novel optogenetic-microdialysis approach, which allows the measurement of extracellular concentrations of glutamate and dopamine in the striatum upon light-induced stimulation of cortical glutamatergic terminals (4). The study demonstrates that adenosine, acting on A2A receptors localized in cortico-striatal glutamatergic terminals (which form heteromers with A1 and CB1 receptors, 5), constitutes a very significant modulatory factor that controls the tonic glutamate-dependent striatal extracellular concentration of dopamine, because local A2A receptor blockade (by striatal perfusion of a selective A2A receptor antagonist) totally abrogated the local glutamate and dopamine release induced by optogenetic stimulation (4). In relation to D1-ghrelin receptor heteromers, our work in mammalian transfected cells and striatal neurons in cultures has demonstrated a very significant functional role of the truncated ghrelin receptor GHS-R1b (6). Ghrelin acts on the growth hormone secretagogue (GHS) receptor or GHS-R1a. Cells expressing GHS-R1a also express GHS-R1b, a truncated variant of GHS-R1a lacking the transmembrane domains 6 and 7. Ghrelin does not bind to and therefore does not signal through GHS-R1b, which until recently was suggested to only exert a dominant-negative effect on the trafficking and signaling of GHS-R1a. Our study demonstrates that GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling, but also the ability of GHS-R1a to form oligomeric complexes with the D1 receptor, promoting profound qualitative changes in ghrelin-induced signaling (6). We are now evaluating the possibility than the dopamine D1-like receptor subtype D5, which is expressed by mesencephalic dopaminergic neurons can also form the same type of oligomers with GHS-R1a and GHS-R1b receptors. In relation to D4 receptors, their variants and heteromers, we have started by re-evaluating the different functional differences of their variants using a novel bioluminescence resonance energy transfer (BRET) assay that measures G protein activation by measuring ligand-induced BRET changes between sensors fused in different G protein subunits (7). The study brought several unexpected findings. The Gi/o-coupled dopamine D2-like receptor family comprises three subtypes: D2 receptor, with short and long isoform variants, D3 receptor and D4 receptor, with several polymorphic variants. Previous studies had suggested that, in addition to dopamine, norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2 or D3 receptors had not been demonstrated. We performed a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2 isoforms and the most common D4 polymorphic variants. We founded that norepinephrine acted as a potent agonist for all D2-like receptor subtypes. For both dopamine and norepinephrine, differences depended on the Gi/o protein subunit involved. Furthermore, the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants (7). These results also support our previous hypothesis about functional differences among D4 receptor variants depending on receptor heteromerization. We are now working on this working hypothesis.